This invention relates to a lytic diluent and a method for rapid lysing of red blood cells in whole blood for a differential determination of lymphoid and myeloid populations of leukocytes, and measurement of hemoglobin, particularly in automatic particle counting systems.
Much effort has been devoted to the development of satisfactorily automated leukocyte differential systems, and several types of instruments are now being used to replace the slow, and error prone manual differential counts. However, a need exists for reagent systems which will be easily adaptable to automatic blood counting instruments. In particular, it is desirable to develop reagents and methods for use with the Coulter Counter.RTM. Model S Plus automated blood counter, manufactured by Coulter Electronics, Inc. of Hialeah, Fla., which will enable the Coulter volume data accumulated on a Coulter Channelyzer.RTM., to discriminate two populations of leukocytes: (1) a lymphoid (lymphocytes) population, and, (2) a myeloid (neutrophils, monocytes, eosinophils, and basophils) population. This information, even though not a complete differential, gives valuable screening data. As a screening tool for spotting abnormal leukocyte situations, such as infection, leukemia, and possibly immature leukocytes, this lytic diluent provides valuable information not contained in ordinary blood counts. Abnormal situations flagged out by this method would require further study to obtain information of diagnostic significance. The two-population information can be used to monitor the process.
A report of previous work with saponin lysed blood showed that red blood cells could be lysed completely, leaving leukocytes in good condition for electronic counting when (1) the saponin concentration is carefully controlled, and (2) lysing is terminated by glutaraldehyde fixation or stabilization with albumin, cold temperature and dilution.
A method of obtaining leukocyte distributions, using about one-half the concentration of detergent as Lyse S.RTM., results in poorly separated populations [J. M. England, et. al, Lancet, Mar. 1, 1975 page 492; J. M. England et. al, Lancet, May 22, 1976; J. M. England, et. al, J. Clin. Path., 27, 623 (1974); P. A. Wycherly and M. J. O'Shea, J. Clin. Path., 31, 271 (1978).] Experiments showed that this Lyse S.RTM. at half concentration was too powerful and that the larger myeloid population was rapidly shrinking in volume during the data accumulation stage, resulting in very severe overlapping of the two populations. The degree of overlap was very dependent on how rapidly the sample could be counted. The lymphoid-myeloid ratio was calculated using a complex curve-fitting routine.
D'Angelo et. al, J. Clin. Path., 38 No. 6, 658-662 studied the use of Cetrimide.RTM. with and without citrate-saline solution as a practical diluent for electronic white cell counts, but without differentiation of lymphoid-myeloid populations.
In U.S. Pat. No. 3,874,852 (1975), Hamill to Coulter Diagnostics, Inc., a formula is included for a composition containing quaternary ammonium salt detergent and cyanide to be employed as a lysing and chromagen-forming reagent for obtaining a single volume leukocyte count and hemoglobin determination in the Coulter Counter.RTM. Model S. This composition is used in alkaline solution since in acid solution potassium cyanide is converted into hydrogen cyanide gas which is evolved causing loss of the cyanide ion from the reagent, as a poisonous gas.
Further investigation was required to use quaternary ammonium salts as lysing agents for obtaining the two-population leukocyte count.
The Coulter Counter.RTM. Model S Plus automated blood counter requires the lyse to be completed within a few seconds of contact with whole blood. The lyse must degrade and digest the red blood cell membrane to a point where the membrane debris will have a small enough volume to be cleanly separated from the leukocytes. For the Coulter Counter.RTM. Model S Plus system the leukocyte volume distribution needs to be completely stable for at least one minute. It is very desirable, however, to have the leukocyte distribution stable for longer periods, for example, several minutes or up to one to two hours, so that the distribution is unchanged during counting, or so that manual dilutions can be examined with a semi-automated device. Also, with longer periods, finger stick blood could be lysed and held for subsequent analysis. The lytic diluent should give good results both on freshly drawn blood, and blood that has been stored for 24 hours or more.
To achieve a simple and accurate analysis of the percentage of lymphoid cells and myeloid cells in blood, the two populations of leukocytes should be cleanly separated such that the valley between the red cell debris and the lymphoid peak is very close to the baseline of the channel, and sufficiently broad to allow setting a fixed threshold channel from which the lymphoid cells can be counted. Likewise the valley between the lymphoid and myeloid peaks must allow for setting a fixed channel number dividing the populations on all blood samples without problems caused by overlap of the populations. Curve fitting programs, and variable valley settings which reduce the ease and accuracy of the results should be avoided.
In addition to allowing the identification of the leukocytes, the lytic diluent must permit formation of a chromagen which gives a satisfactory correlation with the concentration of hemoglobin in the whole blood sample.